Goat anti mouse igg antibody conjugated with horseradish peroxidase

The cells were lysed using radioimmunoprecipitation assay (RIPA) buffer (Cat# 16488-34, Nacalai Tesque) containing protease inhibitors. Proteins were separated using SDS-PAGE and transferred to Immobilon-P membranes (Cat# IPVH00010, EMD Millipore, Billerica, MA, USA). After blocking with 5% milk in PBS-T for 1 h, membranes were incubated with primary antibodies at 4°C overnight. Membranes were then washed with PBS-T and incubated with secondary antibodies for 2 h. Membranes were washed again with PBS-T and immunoblot signals were developed using EzWestLumi plus (Cat# WES-7120, ATTO, Tokyo, Japan) and recorded using an ImageQuant LAS4000 mini-image analyzer (GE Healthcare Japan, Tokyo, Japan). The antibodies used were as follows: anti-spike antibody 1A9 (Cat# GTX632604, GeneTex, CA, USA), anti-cleaved spike (Ser 686) antibody (Cat# 84534, Cell Signaling Technology, Massachusetts, USA), anti-GAPDH 3H12 (Cat# 171-3, Medical & Biological Laboratories, Aichi, Japan), goat anti-mouse IgG antibody conjugated with horseradish peroxidase (Cat# SA00001-1, Proteintech Group, Rosemont, USA), donkey anti-rabbit IgG antibody conjugated with horseradish peroxidase (Cat# NA934, Cytiva, Tokyo, Japan).

Wells of 96-well microtiter plates were coated with 1 μg/mL purified recombinant SARS-CoV-2 Trimeric Spike ECD and RBD (Spike WT; Cat# 40589-V08H4, Spike BA.1; Cat# 40589-V08H26, Spike BA.4/5; Cat# 40589-V08H32, Spike BQ.1; Cat# 40589-V08H41, Spike BA.2.75; Cat#40589-V08H36, RBD BQ.1; Cat# 40589-V08H143, Sino Biological, Beijing, China) at 4°C overnight. Plates were blocked with 1% BSA in PBS-T for 1 h. Antibodies were diluted in blocking buffer, added to the wells, and incubated for 2 h at room temperature. For peptide competition assay, CvMa-62 was preincubated with epitope peptide at 37°C for 30 min. The bound antibodies were detected using goat anti-mouse IgG antibody conjugated with horseradish peroxidase (Proteintech Group) and TMB substrate (Cat# 34028, 1-Step TMB ELISA Substrate Solutions, Thermo Fisher Scientific, Carlsbad, CA). Color development was monitored, 2 M sulfuric acid was added to stop the reaction, and the absorbance was measured at 450 nm using a multi-mode plate reader SpectraMax iD3 (Molecular Devices, CA, USA).